Fig. 2: Interaction of EXO1 with MLH1 has only a minor impact on the nuclease of MutSγ-MutLγ. | Nature Communications

Fig. 2: Interaction of EXO1 with MLH1 has only a minor impact on the nuclease of MutSγ-MutLγ.

From: EXO1 promotes the meiotic MLH1-MLH3 endonuclease through conserved interactions with MLH1, MSH4 and DNA

Fig. 2: Interaction of EXO1 with MLH1 has only a minor impact on the nuclease of MutSγ-MutLγ.

a Multiple sequence alignment of a conserved region of EXO1 from various species generated with MAFFT and visualized in Jalview using ClustalW color coding. Dashes (−) represent gaps in the alignment. b AlphaFold2 model of the complex between the C-terminal domains of MutLγ and EXO1. Top, C-terminal domains of MutLγ are shown in light and dark blue for MLH3 and MLH1, respectively, and the interacting regions of EXO1 are shown in pink. Bottom left, residues interacting with MLH1 in the 390–410 stretch of EXO1 (centered around I403). Bottom right, conserved MIP motif (R-S-R-F-F, residues 503–507) of EXO1 interacting with MLH1. Asterisk indicates the residue mutated in this study. c Cartoons of the primary structure of EXO1. The N-terminal catalytic domain is shown in purple, and the C-terminal tail is shown in magenta. Mutations of EXO1 residues predicted to be involved in the interaction with MutLγ are highlighted. MIP mutations, F506A and F507A mutations within the MIP motif. The nuclease-dead D173A mutation is indicated for reference. d Protein interaction assays of MutLγ and EXO1 variants. MutLγ (FLAG-MLH1 and MBP-MLH3, bait) was immobilized using an anti-MLH1 antibody, and EXO1-FLAG variants (prey) were subsequently added. MIP mutations, F506A and F507A mutations within the MIP motif. Top, a schematic. Bottom, a representative experiment. Right, quantification of interaction relative to EXO1 (D173A), normalized to MLH1, averages shown, n = 4 independent experiments; error bars, SEM; two-tailed t-test. e Nicking assays testing the effect of EXO1 (D173A) variants on the stimulation of MutLγ-MutSγ complex in the presence of 0.6 mM Mn2+ (without RFC-PCNA) (left); and in the presence of 0.3 mM Mg2+ and RFC-PCNA (right). MIP mutations are F506A and F507A within the MIP motif. Nuclease-dead yeast Exo1 (D173A) was used in lanes 7 and 14 as a negative control. Top, quantification; averages shown, n = 4 (experiments with Mn2+) and n = 3 (experiments with Mg2+); error bars, SEM; two-tailed t-test. Bottom, representative gels. Source data are provided as a Source Data file.

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